Matrix protein compositions for modulating immune response

ABSTRACT

The present invention relates to the use of a preparation of an active enamel matrix substance, such as an amelogenin, for the manufacture of a pharmaceutical composition for modulating an immune response. The composition can be used in preventing and/or treating a condition or disease in a mammal that is characterised by said mammal presenting an imbalance in its native immune response to an internal and/or external stimuli, i.e. wherein at least a part of said mammal&#39;s immune system is stimulated non-discriminatingly, reacts hypersensitively to said immunogen, or fails to react to said stimuli. Said condition can typically either be systemic or local, such as a systemic and/or post-traumatic whole-body inflammation or an autoimmune disease.

FIELD OF THE INVENTION

[0001] The present invention relates to the use of a preparation of anactive enamel matrix substance, such as an amelogenin, a processedamelogenin product, or a metabolite thereof, for the manufacture of apharmaceutical composition for modulating an immune response. Thecomposition can be used in preventing and/or treating a condition ordisease in a mammal that is characterised by said mammal presenting animbalance in its native immune response to an internal and/or externalstimuli. Said condition can typically either be systemic or local, suchas a systemic and/or post-traumatic whole-body inflammation or anautoimmune disease.

BACKGROUND OF THE INVENTION

[0002] The normal function of the immune system is to protect the bodyby attacking and destroying foreign microorganisms, such as bacteria andviruses. Under normal conditions, these would be recognised as invadersand thus be rendered harmless.

[0003] Responsible for a subject's immune response is the lymphoidsystem; meaning the assemblage of lymphocytes, their precursors andderivatives, and all supportive cells. Under abnormal conditions orimbalance of the immune system, it fails to react and destroy theforeign microorganisms, mistakes own cells and tissues as foreign andattacks them, or is activated inappropriately in a hypersensitivemanner.

[0004] An immune response to an antigenic agent, be it a foreign antigenor an auto-antigen, is generally characterised by the production ofantibodies by B lymphocytes, and destruction of any cells displayingthose antigens by T lymphocytes or natural killer (NK) cells. Defects inB or T lymphoid cells, however, may result in the development ofimmunodeficiency diseases or the impairment of the immune responsefunction. The immune deficiency or defect may be congenital, i.e. causedby a mutation in a gene, or it may be acquired, e.g. through a viralinfection or as a result of the ageing process. The thus produced defectmay or may not be fatal, depending on the stage of stem cell orlymphocyte differentiation at which it occurs.

[0005] In addition to an antigen, lymphocytes need lymphokines for fullactivation. E.g., the T-helper cells do not proliferate unless theyreceive signals from macrophages, and T-cytotoxic cells as well asB-cells depend on signals from T-helper cells and probably also frommacrophages for their full development. An adequate immune responseneeds the coordinated activities of several cell types, which means thatmodulating the expression or activity of lymphokines potentially has abeneficial effect on adverse immune responses.

[0006] After shocks, burns, extensive surgical procedures or traumaticinjury, functions of the immune system can be over-stimulated within asystemic, non-discriminant, excessive whole-body inflammation, whereasother functions are dramatically paralysed. When the immunological hostresponse is thus uncontrolled, the unbalanced states of cellularactivation that are observed lead to consecutive clinical states. Suchstates can be acute-phase response, whole-body inflammation, anergy,sepsis, infection or organ failure and may finally lead to multipleorgan failure. Multiple organ failure or dysfunction is the most commoncause for death in intensive care units. As of today, therapy directedto prevent or improve uncontrolled immune response has not been able todramatically alter the outcome.

[0007] Other examples for unbalanced situations are when the immunesystem attacks its own body. These situations differ in the origin ofthe antigen that triggers the attack and the mechanisms andmanifestations of the attack. These reactions are often referred to asallergy, delayed-type hypersensitivity, allograft, and autoimmunereactions, respectively.

[0008] Anergy is another unbalanced condition, in which the body failsto react to an injected allergen or antigen. Anergy is thought to be theresult of intercellular signalling after interaction between T-cellreceptor (TCR) and peptide-presenting major histocompatibility complex(MHC) antigen in the absence of a “costimulatory” signal. Thiscostimulatory signal is normally provided on the cell surface ofantigen-presenting cells (APCs).

[0009] The immune system modulates its function both locally andsystemically. E.g. delayed-type hypersensitivity can manifest itselfeither locally in a characteristic skin reaction, or in a systemicreaction in which large quantities of the antigen enter the bloodstreamand which is characterised by fever, malaise, pains in the joints andreduction of the number of circulating lymphocytes. Inflammation isgenerally defined as a local response to cellular injury, whereas wholebody inflammation or multiple organ failure is a systemic inflammation.

[0010] Posttraumatically, monocytes and macrophages are immediatelyhyperactivated to excessively release proinflammatory cytokines. Thisexcessive release results in the development of whole body inflammation,followed in most patients by substantial paralysis of cell function.After 3 to 5 days this state is overcome with newly recruitedmonocytes/macrophages that probably lack the full spectrum of activity,though, because they are immature. In addition, patients can alsoundergo an anti-inflammatory phase (the compensatory anti-inflammatoryresponse syndrome), and at times a mixed response with both pro-andanti-inflammatory components (the mixed antagonistic response syndrome)is observed.

[0011] The posttraumatic impact on the balance of cell-mediated immuneregulation is thus primarily caused by a simultaneous attack on themonocyte/macrophage lineage and T cells, causing disintegration of theintact cell interaction. Traumatic stress not only effects the capacityof adequate and specific performance of each cell type; it also effectscontrol capacity and modulatory surveillance that themonocyte/macrophage lineage and T cells normally posses for each otherwithin a number of regulatory loops. This loss of regulatory functionoccurs instantaneously at the moment of injury.

[0012] Known inflammatory cytokines to cause the above-described initialsystemic hyperinflammation are e.g. Tumour necrosis factor-a(TNF-alpha), Interleukin-1 (IL-1), IL-6, and Interferon gamma, whichmight act synergistically with TNF-alpha. Clinical trials aimed atdown-regulating these mediators, using e.g. antibodies againstendotoxin, TNF-alpha, antagonists of IL-1, or platelet activatingfactor, have so far been uniformly disappointing. Not only have theseagents been found to have no regulating effect, they even increasedmortality.

[0013] Furthermore, under stressful conditions, the monocyte/macrophagelineage's are easily triggered to produce and release prostaglandin E₂(PGE₂), which is probably the most powerful endogenous immunesuppressant. PGE₂ is an inhibitor of T cell mitogenesis, IL-2 receptorexpression and IgM antibody synthesis by B cells. Via intracellularelevation of cAMP levels, PGE₂ also negatively controls themonocyte/macrophage lineage synthesis of TNFα and IL-1. PGE₂ can inhibitthe synthesis of TH₁ cytokines (IL-2, IFNγ) but not the synthesis ofIL-4 by TH₂ cells. Thus PGE₂ secretion may tip the balance in favour ofa TH₂ type response, leading to a switch in B cell production from IgMto IgG1 and IgE. Tipping the balance thus influences the development ofa TH₁ or a TH₂-dominated response.

[0014] Traditional reagents and methods used to regulate a patient'simmune response often result in unwanted side effects. For example,immunosuppressive reagents, such as cyclosporine A, azathioprine andprednisone are used to suppress the immune system of a patient with anautoimmune disease or patients receiving transplants. Such reagents,however, suppress a patient's entire immune response, thereby cripplingthe ability of the patient to mount an immune response againstinfectious agents not involved in the original disease. Due to suchharmful side effects and the medical importance of immune regulation,reagents and methods to regulate specific parts of the immune systemhave been the subjects of study for many years.

[0015] Enamel matrix proteins, present in the enamel matrix, are mostwell known as precursors to enamel. Prior to cementum formation, enamelmatrix proteins are deposited on the root surface at the apical end ofthe developing tooth-root. The deposited enamel matrix is the initiatingfactor for the formation of cementum. Again, the formation of cementumin itself is associated with the development of the periodontal ligamentand the alveolar bone. As shown by the present inventors prior to thepresent invention, enamel matrix proteins can therefore promoteperiodontal regeneration through mimicking the natural attachmentdevelopment in the tooth (Gestrelius S, Lyngstadaas S P, Hammarstrom L.Emdogain—periodontal regeneration based on biomimicry. Clin Oral Invest4:120-125 (2000).

[0016] The enamel matrix is composed of a number of proteins, such asamelogenin, enamelin, tuft protein, proteases, and albumin. Amelogenins,the major constituent of the enamel matrix, are a family of hydrophobicproteins derived from a single gene by alternative splicing andcontrolled post secretory processing. They are highly conservedthroughout vertebrate evolution and demonstrate a high overall level ofsequence homology among all higher vertebrates examined (>80%). In fact,the sequences of porcine and human amelogenin gene transcript differonly in 4% of the bases. Thus, enamel matrix proteins, although ofporcine origin, are considered “self” when encountered in the human bodyand can promote dental regeneration in humans without triggeringallergic responses or other undesirable reactions.

[0017] Enamel matrix proteins and enamel matrix derivatives (EMD) havepreviously been described in the patent literature to be able to inducehard tissue formation (i.e. enamel formation, U.S. Pat. No. 4,672,032(Slavkin)), binding between hard tissues (EP-B-0 337 967 and EP-B-0 263086) and wound healing, such as of skin and mucosa (WO 99/43344).

SUMMARY OF THE INVENTION

[0018] The present invention is based on the surprising finding thatactive enamel substances have the ability to modulate immune mechanismsof mammals. Immunomodulation herein being defined as an increase ordecrease in the level of immune response brought about by variousspecific and non-specific means.

[0019] A preferred embodiment of the present invention thus comprisesthe use of an active enamel substance for the manufacture of apharmaceutical composition for the prevention and/or treatment of acondition in a mammal, wherein said condition is characterised by animbalance in the immune response of said mammal to an immunogen, such asan external or internal stimulatory factor.

[0020] An “imbalance in the immune response” can occur after shocks,burns, extensive surgical procedures and/or traumatic injury. The termdescribes a situation/condition, wherein one or more functions of theimmune system can be either over-stimulated within a systemic,nondiscriminant, excessive whole-body inflammation, whereas otherfunctions can simultaneously or independently be under-stimulated. Theterm “imbalance in the immune response” describes a situation/conditionin which the immunological host response is uncontrolled, and whereinthe unbalanced states of cellular activation that are observed lead toconsecutive clinical states. Such states can be acute-phase response,whole-body inflammation, anergy, shock, sepsis, infection and/or organfailure and may finally lead to multiple organ failure.

[0021] Other examples for “imbalance in the immune response” aresituations/conditions wherein the subject's inert immune system attacksits own body. These situations differ in the origin of the antigen thattriggers the attack and the mechanisms and manifestations of the attack.These reactions are often referred to as allergy, delayed-typehypersensitivity, allograft, and autoimmune reactions, respectively.

[0022] The term “imbalance in the immune response” can describe bothlocal and systemic situations, as described above. E.g. Inflammation isgenerally defined as a local response to cellular injury, whereas wholebody inflammation or multiple organ failure are examples of systemicinflammation.

[0023] In the present context, the term “an immunogen” is used todescribe any factor that is able to elicit an immune response. This cane.g. be a toxin, fungus, parasite, virus, bacteria, pollen, dust,pollution particle, cytokine, hormone, stress, surgery, burn,transplanted organ or blood, tissue graft, shock, trauma, or a non-selfor a self antigen.

[0024] Immunological hypersensitivity reactions are immune responseswhich lead to tissue damage or impaired body function. They areclassified into five types (I-V), according to the mechanism of thetissue damage, they are involved in many important human diseases anddisorders, such as anaphylaxis, asthma, food allergies, myastheniagravis and systemic lupus erythematosus.

[0025] In one embodiment of the present invention, an active enamelsubstance is used for the manufacture of a pharmaceutical compositionfor the prevention and/or treatment of a condition in a mammal, whereinat least a part of the immune system reacts hypersensitively to saidimmunogen. In a preferred embodiment, at least a part of the immunesystem of said mammal is thus stimulated non-discriminatingly.

[0026] The conditions which may benefit from the invention compriseconditions wherein the imbalance of the immune response of said mammalis either local and/or systemic. In the present context systemic isdefined as relating to or common to a system, i.e. affecting the bodygenerally, in contrast to being local. Said condition can e.g. be anauto-immune disease selected from the group consisting of rheumatoidarthritis, diseases of the skin, allergy, sclerosis, arteriosclerosis,multiple sclerosis, asthma, psoriasis, lupus, systemic lupuserythematosis, diabetes mellitus, myasthenia gravis, chronic fatiguesyndrome, fibromyalgia, chronic fatigue syndrome, Crohn's disease,Hashimoto's thyroiditis, Grave's disease, Addison's disease, GuillianBarre syndrome and scleroderma.

[0027] In the present context, the term “autoimmune disease” refers to awide variety of over 80 different serious chronic illnesses. Autoimmunediseases are the third major category of illness in the United States,behind cancer and heart disease. Between 75-90% of all autoimmunediseases occur in women and usually within the childbearing years. Inaddition to body organs, autoimmune diseases also affect nerves,muscles, blood, connective tissues, gastrointestinal system and all bodysystems.

[0028] The cause of autoimmune diseases is not fully known orunderstood, however, it is suspected that hormones, heredity andenvironmental factors play a major role in their cause and onset. Thecourses of most of these diseases are individualised and highlyunpredictable. Autoimmune diseases do not represent generalised failuresin the tolerance mechanism, rather they reflect the appearance of aspecific response to a specific self-antigen.

[0029] Other conditions equally envisioned in the present invention arecharacterised as systemic inflammations and/or infections and can beassociated with trauma, burns, surgery, dialysis, shock and/or bacterialor viral infection. In a preferred embodiment of the invention, thesystemic condition is associated with a disease selected from the groupconsisting of extracorporeal membrane oxygenation (ECMO), shock,systemic inflammatory response syndrome (SIRS), sepsis, multi organfailure, rejection of an implant such as organ and/or prostheses, heartand lung bypass surgery, chronic inflammation, asthma and/or stressrelated disease in the lung, bacterial infection, gangrene, soft tissueinfection, and wound infection.

[0030] Several severe trauma, such as major surgery, systemicinflammation, burns, or shock can result in massive impairment ofimmunological reactivity in a patient, the clinical consequence of whichis high susceptibility of the traumatised individual towards serioussystemic infection that can lead to massive tissue destruction and organfailure.

[0031] In one embodiment of the present invention, active enamelsubstances are thus used for the manufacture of a pharmaceuticalcomposition for the prevention and/or treatment of a condition in amammal, wherein at least part of the immune system attacks molecules,cells, or tissues of the organism producing them. Active enamelsubstances are herein used for the manufacture of a pharmaceuticalcomposition for the prevention and/or treatment of a condition in amammal, wherein the condition is characterised as a systemicinflammation and/or infection.

[0032] Within a systemic, nondiscriminant, excessive whole-bodyinflammation, parts of the immune system are stimulated, whereas otherfunctions within the complex of cell-mediated immunity (CMI) aredramatically paralysed. Immune abnormalities in the aftermath of traumaoccur in a sequence of states of cellular activation and within acomplex order of events, that is not yet well understood. One known factis that traumatic stress causes disintegration of the intact monocyte-Tcell interaction, which is associated with profound changes in monocyteregulation and substantial depression of T cell function. In a preferredembodiment of the present invention, active enamel substances are thusused for the manufacture of a pharmaceutical composition for restoringthe balance in the monocyte-T cell interaction of a patient, i.e. forrestoring a healthy monocyte regulation and T cell function.

[0033] In the current context, inflammation is defined as a localresponse to cellular injury that is marked by capillary dilatation,leukocytic infiltration, redness, heat, pain, swelling, and/or loss offunction and that normally serves as a mechanism initiating theelimination of noxious agents and of damaged tissue.

[0034] Active enamel substances are in the present invention used forthe manufacture of a pharmaceutical composition for the modulation ofthe conception of T-helper cell activation in a mammal. The activeenamel substance comprised in the pharmaceutical composition can hereinmodulate the level of expression, release and/or function of eitherinter- and/or intra-cellular signalling substances. The active enamelsubstance comprised in the pharmaceutical composition can alternativelymodulate the level of expression, release and/or function of at leastone cytokine involved in the immune mechanism of said mammal or modulatea cellular signalling cascade involved in the immune mechanism of saidsame mammal.

[0035] Many common and clinically important disease states, such asrheumatoid arthritis, asthma, tuberculosis, leprosy, schistosomiasis,chronic hepatitis, thyroiditis and multiple sclerosis, are examples ofchronic inflammation and its consequences. Chronic inflammation mayresult from failure to eliminate an acute inflammatory irritant, from anautoimmune response to a self-antigen, or may be caused by an innatelychronic irritant of low intensity that persists. It is characterised bysimultaneous inflammation and repair, with recruitment and activation ofmacrophages, lymphocytes and other cells triggered by the coordinatedaction of cytokines and growth factors. A preferred embodiment of thepresent invention comprises the use of an active enamel substance forthe manufacture of a pharmaceutical composition for the treatment and/orprevention of chronic inflammation.

[0036] Chronic inflammation is most appropriately defined in terms ofthe process, in which continuing inflammation and attempted tissuehealing by repair occur simultaneously. Although it is often definedsimply in terms of time course, with lesions of over 6 weeks' durationtraditionally being regarded as chronic, any such definition is entirelyarbitrary. At a microscopic level, chronic inflammation is sometimesdefined in terms of the pattern of cellular response, although this isvariable and not altogether reliable.

[0037] The distinctive features of this process are best appreciated bycomparing it with the acute inflammatory response. In contrast to acuteinflammation, where the host response leads to elimination of theirritant, followed by recovery involving tissue regeneration or repair,chronic inflammation is characterised by inflammation and repairoccurring concurrently, rather than consecutively. Repair is always afeature of chronic inflammation because it is associated with irritantsthat cause destruction of tissue architecture. Repair is typicallyachieved by ingrowth of granulation tissue, which includes macrophages,fibroblasts and new blood vessels.

[0038] Other important distinctions between acute and chronicinflammation relate to the relative balance between exudation andcellular recruitment, as well as the types of cells that predominate inthe inflammatory response. In chronic inflammation there is typically aless pronounced exudative response and increased inflammatory cellularrecruitment, which may be accompanied by local cellular proliferation.In contrast to acute inflammation, which is usually characterised byrecruitment of large numbers of neutrophil leukocytes, the dominantinfiltrating cell in all forms of chronic inflammation is themacrophage. Depending on the nature of the irritant, different profilesof inflammatory mediators and growth factors (collectively referred toas cytokines) are generated locally, giving rise to differentmorphological patterns of chronic inflammation.

[0039] The systemic effects of inflammation are more pronounced inchronic inflammatory diseases and may contribute significantly to theclinical consequences. These systemic effects are largely mediated bycytokines. Whereas the most prominent systemic effects of acuteinflammation are fever and leukocytosis, chronic inflammation is usuallyassociated with fatigue, sleepiness, weight loss and wasting.

[0040] When the condition and/or disease related to in the presentinvention is characterised as an infection, said condition can be causedby a contamination with a bacterium selected from but not limited to thegroup consisting of gram negative microbes and gram positive microbes 1)G+: E.g. Staphylococci and streptococci, which are able to release theendo-toxin peptidoglycan (PepG) that is a specific cell wall componentof G+ microbes. 2) G-: E.g. Coli-forms and other microbes releasinglipopolysaccharides (LPS) from their outer cell envelope duringinfection. The immunological condition is herein caused by an endo-toxinand/or an exo-toxin, produced by said infecting microbe.

[0041] In the present invention, cellular responses in human blood,caused by endo-toxins from Staphylococcus aureus (PepG) and Escherichiacoli (LPS) are effectively modulated by administration of amelogenins,as shown in example 1. These results clearly demonstrate the potentialmodulatory effect of active enamel substances on the immune mechanism inmammals.

[0042] In another experiment, conducted by the inventors and documentedin the present application as example 2, using a pig model for sepsis,the therapeutic potential of enamel matrix derivatives is examined invivo.

[0043] EMD, even delivered by big bolus injections, is in experiment 2shown to be safe to administer systemically in high dosages. Moreover,it is herein proven that systemic injections of enamel matrixderivatives counteract the effect of endotoxin (LPS) in pigs. Generally,it can be concluded that the onset of septic shock in the treated pigsare delayed, compared to sham operated pigs. The delay is dependent ondosage, 1 mg/kg bodyweight only gives a short delay (about 30 minutes)while a dosage 5 mg/kg seems to protect against, or at leastsignificantly delay (>5 hours), the onset of septic shock and therelated multi-organ failures.

[0044] One possible mechanism that could explain the beneficial effectof enamel matrix derivatives on LPS induced shock is that enamel matrixderivatives bind LPS and thus inactivates the toxic effect. Another,equally envisable possibility is that enamel matrix derivatives competewith LPS for the same receptor(s) on e.g. white blood cells,macrophages, osteoclasts and/or mastcells. Thus, a preferred embodimentof the invention relates to the use of an active enamel substance forthe preparation of a pharmaceutical composition, wherein said activeenamel substance inactivates the toxic effect of an endo-toxin and/orexo-toxin produced by a microbe, by binding directly to said endo-toxinand/or exo-toxin, and/or by binding to the cellular receptor for saidendo-toxin and/or exo-toxin.

[0045] A third and highly probable possible mechanism is that enamelmatrix derivatives modulate the immune response directly by regulating,in a favourable way, the expression cascade of cytokines and signalmolecules that are major players in the LPS induced septic shock. Basedon previous ex vivo experiments not described in the presentapplication, it seems reasonable to ascribe at least part of the enamelmatrix derivatives' effect to the latter mechanism. Furthermore, theblood analyses performed also suggest that enamel matrix derivativeshave a direct effect on cytokine expression in white blood cells. Theeffect appears to be specific, having a strong effect on TNF and IL-6,but non on IL-1β. Finally, it is well possible that all the mechanismsproposed above are involved, and that it is the combination of theseeffects that give the beneficial effect of enamel matrix derivatives inthis model.

[0046] The delay of the onset of septic shock, reported in example 2,has significant clinical value. In one envisioned embodiment of theinvention, the delay is directly related to the amount of “free” LPS inthe blood, and enamel matrix derivatives will thus be delivered as anadjunct to antibiotics in severe bacteraemia and infections likemeningitis, to delay and/or reduce the effect of toxins. Such treatmentwill be crucial for the survival of the patient in the critical hours,following the start of antibiotic treatment.

[0047] In another, equally preferred embodiment, the effect of enamelmatrix derivatives is caused by direct modulation of the inflammatoryresponse, and thus enamel matrix derivatives might be used in a widearray of clinical situations in which shock is a possible complication.This includes all major surgery, transplantations, trauma, ECMO, acutedialysis, severe infections, severe allergies and many more.

[0048] Without the intention to limit the scope of the presentinvention, one theoretical explanation for the mechanism by which anactive enamel substance as described herein is able to modulate theimmune system of a mammal, is its interaction with a cell surfacereceptor and/or a cell surface structure, selected from the groupconsisting of ICAM, HLA antigen, Interleukin 1 receptor, Interleukin 2receptor, Interleukin 3 receptor, Interleukin 4 receptor, Interleukin 5receptor, Interleukin 6 receptor, Interleukin 7 receptor, Interleukin 8receptor, Interleukin 9 receptor, Interleukin 10 receptor, Interleukin11 receptor, Interleukin 12 receptor, Interleukin 13 receptor,Interleukin 14 receptor, Interleukin 15 receptor, Interleukin 16receptor, Interleukin 17 receptor, Interleukin 18 receptor, IFNαreceptor, IFNβ receptor, IFNΔ receptor, IFNγ receptor, IFNΩ receptorPDGF receptors, TGF receptors, TNF receptor, EGF receptor, the CD44variants and the integrine receptor family.

[0049] Cell surface receptors are well known mediators of cellularcommunication, when activated by their respective signalling molecule,the ligand. The cell surface receptor binds the ligand with high or lowaffinity and converts this extracellular event into one or moreintracellular signals that alter the behaviour of the target cell.Active enamel substances may thus be able to modulate the immune systemby altering the behaviour of the target cell through a similar manner.Thus, in one embodiment of the present invention, active enamelsubstances activate a cell surface receptor, thereby inducing a receptorspecific intercellular signalling cascade and altering the expression ofone or more genes leading to an alteration of the behaviour of saidcell.

[0050] In another embodiment of the present invention, an active enamelsubstance can activate a cell surface receptor that operates directly asan enzyme, a so called catalytic receptor, an example being atransmembrane protein with a cytoplasmic domain that functions as atyrosine kinase.

[0051] In yet another preferred embodiment of the present invention, anactive enamel substance activates a G-protein-linked surface receptorthat indirectly activates or inactivates a separate plasma membranebound enzyme or an ion channel. The interaction between the receptor andthe enzyme and/or ion channel is typically mediated by a third proteinsuch as a G protein (GTP-binding regulatory protein), which activatesintracellular mediators, such as but not limited to cyclic AMP, InsP3and Ca^(2+.)

[0052] Cytokines like Interleukins, IFNγ and TNFα all play a criticalrole in the regulation of the immune response. Particular interest hasbeen centred on cytokine gene regulation as part of T celldifferentiation into the TH₁ and TH₂ subsets. The effect of a cytokinecan e.g. be mediated through its specific receptor that initiates asignal transduction pathway, which can in itself result in the releaseof cytokines in a feedback mechanism.

[0053] As demonstrated in example 1, amelogenin has modulatory effectson the cytokine release and regulation of inflammatory surface receptorsin response to bacterial cell wall products. An embodiment of thepresent invention thus encloses the use of an active enamel substancefor the manufacture of a pharmaceutical composition for modulating animmune response by mediating a signal through a cytokine receptor, suchas IL-2R, IL-4R, IL-10R, IFNγR and TNFαR. Alternatively, as the releaseof cytokines is regulated by the level of their expression, an activeenamel substance can also modulate an immune response by initiating anup-and/or down-regulation of the expression level of one or morecytokine(s) in a mammal, thereby modulating the release and/or functionof one or more cytokine(s).

[0054] In an especially preferred embodiment of the present invention,an active enamel substance mediates a regulation of an immune responsethrough a TNFαR. TNFα receptors are expressed on all somatic cell typeswith the exception of erythrocytes. Two receptors of 55 kDa (TNF-R1;also designated:CD120a, also referred to as TNF receptor superfamilymember 1A (TNF-RSF1A) and 75 kDa (TNF-R2; also designated: CD120b, alsoreferred to as TNF receptor superfamily member 1B (TNF-RSF1B) have beendescribed. TNFαR is related to the low affinity receptor of NGF and tohuman cell surface antigen CD40 and is expressed strongly on stimulatedT-cells and B-lymphocytes. A third receptor subtype is expressed innormal human liver. It binds TNFα but not TNFβ.

[0055] Differential effects of the two receptor subtypes have beenfound. It appears that engagement of the p55 receptor specifically leadsto the induction of the cellular adhesion molecules ICAM-1, E-selectin,V-CAM-1, and CD44, while engagement of both the p55 and the p75 receptorinduces expression of α-2 integrine. Apart from the membrane-boundreceptors, several soluble proteins that bind TNF have been described.These proteins of approximately 30 kDa, called T-BP-1 and T-BP-2 (tumournecrosis factor binding proteins), are derived from the TNF-bindingdomain of the membrane receptor. They probably function as physiologicalregulators of TNF activities by inhibiting binding of TNF to itsreceptor.

[0056] The biological activities of IL-2 are mediated by a membranereceptor that is expressed almost exclusively on activated, but not onresting, T-cells. IL-5 and IL-6 modulate the expression of the IL-2receptor. In another preferred embodiment of the present invention, anactive enamel substance is envisioned that mediates a regulation of animmune response through an IL-2R.

[0057] Three different types of IL-2 receptors are distinguished thatare expressed differentially and independently. The high affinity IL-2receptor constitutes approximately 10 percent of all IL-2 receptorsexpressed by cells. This receptor is a membrane receptor complexconsisting of the two subunits IL-2R-alpha (TAC antigen=T-cellactivation antigen; p55) and IL-2R-beta (p75; new designation:CD122).p75 is expressed constitutively on resting T-lymphocytes, NK-cells, anda number of other cell types while the expression of p55 is usuallyobserved only after cell activation. p75 is involved in IL-2-mediatedsignal transduction. In addition, the IL-2 receptor is associated with anumber of other proteins (p22, p40, p100) which are thought to beinvolved in mediating conformational changes in the receptor chains,receptor-mediated endocytosis, and further signal transductionprocesses. One of the identified proteins is the 95 kDa cell adhesionmolecule ICAM-1 which probably focuses IL-2 receptors at regions ofcell-to-cell contacts and thus may mediate paracrine activities, forexample, during IL-2-mediated stimulation of T-cells. Another proteinassociated with p75 is a tyrosine-specific protein kinase called Ick,other kinases may be associated also with IL-2 receptors. Two suchkinases, called fyn and lyn, have been identified. In addition, IL-2receptor signalling may be mediated also by vav. A third 64 kDa subunitof the IL-2 receptor, designated gamma, has been described recently.This subunit is required for the generation of high and intermediateaffinity IL-2 receptors but does not bind IL-2 by itself. The gammasubunit of the IL-2 receptor has been shown recently to be a componentof the receptors for IL-4 and IL-7. It is probably also a component ofthe IL-13 receptor.

[0058] Activated lymphocytes continuously secrete a 42 kDa fragment ofthe TAC antigen. This fragment circulates in the serum and plasma andfunctions as a soluble IL-2 receptor (sIL-2R). The concentrations ofthis soluble receptor varies markedly in different pathologicalsituations.

[0059] In another preferred embodiment of the present invention, anactive enamel substance activates the IFNγR. A number of bindingproteins with molecular masses between 70 and 160 kDa have beendescribed for IFNγR. They are expressed on all types of human cells withthe exception of mature erythrocytes. The receptor expressed inmonocytes and other haematopoietic cells has a molecular mass of 140kDa. A 54 kDa protein is observed in other cell types.

[0060] The biological activities of IL-4 are mediated by the IL-4receptor (IL-4R). In a preferred embodiment of the present invention, anactive enamel substance activates the IL-4R. The extracellular domain ofthe IL-4 receptor is related to the receptors for Epo, IL-6 and the betachain of the IL-2receptor. It has been given the nameCD124. Two forms ofthe receptor have been described, one of which is secreted. The secretedreceptor only contains the extracellular IL-4 binding domain and iscapable of blocking IL-4 activities. An IL-4 binding protein (IL-4-BP)that binds IL-4 with the same affinity as the IL-4 receptor has beenshown to be a soluble IL-4 receptor variant. Soluble receptors probablyfunction as physiological regulators of cytokine activities byinhibiting receptor binding or by acting as transport proteins. Themechanisms of IL-4-mediated intracellular signal transduction arelargely unknown. IL-4 influences intracellular calcium levels and alsothe metabolism of inositol phospholipids and protein kinase C.

[0061] In humans, IL10 is produced by activated CD8⁺ peripheral bloodT-cells, by Th₀, Th₁-, and Th₂-like CD4⁺ T cell clones after bothantigen-specific and polyclonal activation, by B-cell lymphomas, and byLPS-activated monocytes and mast cells. IL-4 and IL10 inhibit thesynthesis of IL10 by monocytes. In a preferred embodiment of the presentinvention, an active enamel substance activates the IL-10 receptor(IL-10R). The murine IL-10 receptor has been cloned. This receptor is aprotein of approximately 110 kDa that binds murine IL-10 specifically.This receptor is structurally related to receptors for IFN.

[0062] The effect of enamel matrix derivatives on peripheral lymphocytesshowed that the porcine amelogenin derivative Emdogain® induces a slightincrease of the proliferation of CD25/CD4 positive lymphocytes in vitro,and a concomitant decrease of CD19 positive B-lymphocytes (Petinaki, E.,S. Nikolopoulos, and E. Castanas. 1998. Low stimulation of peripherallymphocytes, following in vitro application of Emdogain. J ClinPeriodontol. 25:715-20). The authors could not demonstrate anysignificant influence on immunoglobulin and/or cytokine (IL-2 and IL-6)production, though.

[0063] Interestingly, the inventors have observed that various cellcultures of fibroblasts (embryonic, dermal, derived from the periodontalligament, fish fin or bird skin), produce twice as much transforminggrowth factor (TGF-β1) when stimulated with EMDOGAIN® (BIORA AB,SWEDEN), compared to non-stimulated cultures. These findings support theconcept of the potential modulatory influence of active enamelsubstances on imbalanced immune responses.

[0064] Enamel matrix is a precursor to enamel and may be obtained fromany relevant natural source, i.e. a mammal in which teeth are underdevelopment. A suitable source is developing teeth from slaughteredanimals such as, e.g., calves, pigs or lambs. Another source is e.g.fish skin.

[0065] Enamel matrix can be prepared from developing teeth as describedpreviously (EP-B-0 337 967 and EP-B-0 263 086). The enamel matrix isscraped off and enamel matrix derivatives (EMD) are prepared, e.g. byextraction with aqueous solution such as a buffer, a dilute acid or baseor a water/solvent mixture, followed by size exclusion, desalting orother purification steps, alternatively followed by freeze-drying.Enzymes may alternatively be deactivated by treatment with heat orsolvents, in which case the derivatives may be stored in liquid formwithout freeze-drying.

[0066] As an alternative source of the enamel matrix derivatives orproteins one may also use generally applicable synthetic routes, wellknown to a person skilled in the art, or use cultivated eukaryoticand/or prokaryotic cells, alternatively modified by DNA-techniques. Theenamel matrix proteins may thus be of recombinant origin andalternatively genetically modified (see, e.g., Sambrook, J. et al.:Molecular Cloning, Cold Spring Harbor Laboratory Press, 1989).

[0067] In the present context, enamel matrix derivatives, or enamelmatrix protein derivatives (EMD) are derivatives of enamel matrix whichinclude one or several enamel matrix proteins or parts of such proteins,produced naturally by alternate splicing or processing, or by eitherenzymatic or chemical cleavage of a natural length protein, or bysynthesis of polypeptides in vitro or in vivo (recombinant DNA methodsor cultivation of diploid cells, either plant or animal cells). Enamelmatrix protein derivatives also include enamel matrix relatedpolypeptides or proteins. The polypeptides or proteins may be bound to asuitable biodegradable carrier molecule, such as polyamine acids orpolysaccharides, or combinations thereof. Furthermore, the term enamelmatrix derivatives also encompasses synthetic analogous substances.

[0068] Proteins are biological macromolecules constituted by amino acidresidues linked together by peptide bonds. Proteins, as linear polymersof amino acids, are also called polypeptides. Typically, proteins have50-800 amino acid residues and hence have molecular weights in the rangeof from about 6,000 to about several hundred thousand Dalton or more.Small proteins are called peptides or oligopeptides.

[0069] Enamel matrix proteins are proteins that normally are present inenamel matrix, i.e. the precursor for enamel (Ten Cate: Oral Histology,1994; Robinson: Eur. J. Oral Science, Jan. 1998, 106 Suppl. 1:282-91),or proteins which can be obtained by cleavage of such proteins. Ingeneral, such proteins have a molecular weight below 120,000 Dalton andinclude amelogenins, non-amelogenins, proline-rich non-amelogenins andtuftelins.

[0070] Examples of proteins for use according to the invention areamelogenins, proline-rich non-amelogenins, tuftelins, tuft proteins,serum proteins, salivary proteins, ameloblastin, sheathlin, andderivatives thereof, and mixtures thereof. A preparation containing anactive enamel substance for use according to the invention may alsocontain at least two of the aforementioned proteinaceous substances.Moreover, other proteins for use according to the invention are found inthe marketed product EMDOGAIN® (BIORA AB, Sweden).

[0071] EMDOGAIN® (BIORA AB, S-205 12 Malmö, Sweden) contains 30 mgenamel matrix protein, heated for 3 hours at about 80° C. in order toinactivate residual proteases, and 1 ml Vehicle Solution (PropyleneGlycol Alginate), which are mixed prior to application, unless theprotein and the Vehicle are tested separately. The weight ratio is about80/8/12 between the main protein peaks at 20, 14 and 5 kDa,respectively.

[0072] In general, the major proteins of an enamel matrix are known asamelogenins. They constitute about 90% w/w of the matrix proteins. Theremaining 10% w/w includes proline-rich non-amelogenins, tuftelins, tuftproteins, serum proteins and at least one salivary protein; however,other proteins may also be present such as, e.g., amelin (ameloblastin,sheathlin) which have been identified in association with enamel matrix.Furthermore, the various proteins may be synthesised and/or processed inseveral different sizes (i.e. different molecular weights). Thus, thedominating proteins in enamel matrix, amelogenins, have been found toexist in several different sizes that together form supramolecularaggregates. They are markedly hydrophobic substances that underphysiologically conditions form aggregates. They may carry or becarriers for other proteins or peptides.

[0073] The amelogenins of developing dental enamel are tissue-specificproteins, rich in proline, leucine, histidine and glutamyl residues, andsynthesised by the ameloblast cells of the inner enamel epithelium.These proteins comprise the bulk of the extracellular matrix thatbecomes mineralised with a hydroxyapatite phase to become the matureenamel. Examination of the amino acid sequences of amelogenins from arange of mammals shows a high degree of evolutionary sequenceconservation, suggestive of specialised function. Recently it has beenshown that multiple amelogenin components, observed in the matrix, ariseboth by a sequence of post-secretory proteolytic processing and by theexpression of alternatively spliced mRNAs generated from the amelogeningene(s) that are located on the sex chromosomes. Physico-chemicalstudies of recombinant amelogenins have shown that they undergo aself-assembly process in vitro generating supra-molecular ‘nanosphere’structures, and recent observations in vivo point to a functional rolefor the nanospheres in the ultrastructural organization of the secretoryenamel matrix, conducive to the organised development of the earliestmineral crystallites.

[0074] Recent studies of amelogenin expression have demonstrated thatalternative-splicing of mouse amelogenin RNA generates seven distinctmRNAs, coding for amelogenin proteins from 194 to 44 amino acid residuesin length.

[0075] Other protein substances are also contemplated to be suitable foruse according to the present invention. Examples include proteins suchas proline-rich proteins and polyproline. Other examples of substancesthat are contemplated to be suitable for use according to the presentinvention are aggregates of such proteins, of enamel matrix derivativesand/or of enamel matrix proteins as well as metabolites of enamelmatrix, enamel matrix derivatives and enamel matrix proteins. Themetabolites may be of any size ranging from the size of proteins to thatof short peptides.

[0076] As mentioned above, the proteins, polypeptides or peptides foruse according to the invention typically have a molecular weight of atthe most about 120 kDa such as, e.g., at the most 100 kDa, 90 kDa, 80kDa, 70 kDa or 60 kDa as determined by SDS PAGE electrophoresis.

[0077] The proteins for use according to the invention are normallypresented in the form of a preparation, wherein the protein content ofthe active enamel substance in the preparation is in a range of fromabout 0.005% w/w to 1000/% w/w such as, e.g., about 0.5-99% w/w, about1-95% w/w, about 10-95% w/w, about 10-90% w/w, about 15-90% w/w, about20-90% w/w, about 30-90% w/w, about 40-85% w/w, about 50-80% w/w, about60-70% w/w, about 70-90% w/w, or about 80-90% w/w.

[0078] A preparation of an active enamel substance for use according tothe invention may also contain a mixture of active enamel substanceswith different molecular weights.

[0079] The proteins of an enamel matrix can be divided into a highmolecular weight part and a low molecular weight part, and it has beenfound that a well-defined fraction of enamel matrix proteins possessesvaluable properties with respect to treatment of periodontal defects(i.e. periodontal wounds). This fraction contains acetic acidextractable proteins generally referred to as amelogenins andconstitutes the low molecular weight part of an enamel matrix (cf.EP-B-0 337 967 and EP-B-0 263 086).

[0080] In the present context, the active proteins are not restricted tothe low molecular weight part of an enamel matrix. At present, preferredproteins include enamel matrix proteins such as amelogenin, tuftelin,etc. with molecular weights (as measured in vitro with SDS-PAGE) belowabout 60,000 Dalton but proteins having a molecular weight above 60,000Dalton have also promising properties as candidates for promotingconnective tissue growth.

[0081] Accordingly, it is contemplated that the active enamel substancefor use according to the invention has a molecular weight of up to about40,000 Da such as, e.g. a molecular weight of between about 5,000 Da andabout 25,000 Da.

[0082] By separating the proteins, e.g. by precipitation, ion-exchangechromatography, preparative electrophoresis, gel permeationchromatography, reversed phase chromatography or affinitychromatography, the different molecular weight amelogenins can bepurified.

[0083] The combination of molecular weight amelogenins may be varied,from a dominating 20 kDa compound to an aggregate of amelogenins withmany different molecular weights between 40 and 5 kDa, and to adominating 5 kDa compound. Other enamel matrix proteins such as tuftelinor proteolytic enzymes normally found in enamel matrix can be added andcarried by the amelogenin aggregate.

[0084] In an especially preferred embodiment of the present invention,said active enamel substance used for manufacturing a pharmaceuticalcomposition for the prevention and/or treatment of a condition in amammal, being characterised by an imbalance in the immune response ofsaid mammal to an immunogen, is selected from the group consisting ofany soluble amelogenin based peptide with an amino acid length betweenapproximately 2-13k Da, including a soluble amelogenin based peptidewith an amino acid length between 2-4.5k Da, 3-5.5k Da, 4-6.5k Da,5-7.5k Da, or 5-13k Da, that is comprised in the eluate represented bythe third and/or fourth peak of an HPLC analysis of processed amelogeninas shown in FIG. 2.

[0085] In a preferred embodiment of the present invention, saidprocessed amelogenin product is a 5k Da polypeptide.

[0086] In accordance to the present invention, an active enamelsubstance may be used together with other active drug substances suchas, e.g. anti-bacterial, anti-inflammatory, antiviral, antifungalsubstances or in combination with local chemotherapy, inducers ofapoptosis, growth factors such as, e.g., TGFβ, PDGF, IGF, FGF, EGF,keratinocyte growth factor or peptide analogues thereof. Enzymes—eitherinherently present in the enamel matrix or preparation thereof oradded—may also be used in combination with an enamel matrix, enamelmatrix derivative and/or enamel matrix protein, especially proteases.

[0087] For the administration to an individual (an animal or a human),an active enamel substance and/or a preparation thereof is preferablyformulated into a pharmaceutical composition containing the activeenamel substance and, optionally, one or more pharmaceuticallyacceptable excipients.

[0088] A composition comprising the active enamel substance to beadministered may be adapted for administration by any suitable route,e.g. by systemic administration to a patient through a hose, syringe,spray or draining device. Furthermore, a composition may be adapted toadministration in connection with surgery, e.g. as a systemicadministration by infusion into the blood, lymph, ascites, or spinalfluids, or by inhalation.

[0089] In the following, examples of suitable compositions containingactive enamel substances are given.

[0090] For the administration to an individual (an animal or a human)the substance(s) are preferably formulated into a pharmaceuticalcomposition containing the substance(s) and, optionally, one or morepharmaceutically acceptable excipients.

[0091] The compositions may be in form of, e.g., fluid, semi-solid orsolid compositions such as, e.g., but not limited to dissolvedtransfusion liquids, such as sterile saline, Ringer's solution, glucosesolutions, phosphate buffer saline, blood, plasma or water, powders,microcapsules, microspheres, nanoparticles, sprays, aerosols, inhalationdevices, solutions, dispersions, suspensions, emulsions, mixtures.

[0092] The compositions may be formulated according to conventionalpharmaceutical practice, see, e.g., “Remington: The science and practiceof pharmacy” 20^(th) ed. Mack Publishing, Easton Pa., 2000 ISBN0-912734-04-3 and “Encyclopaedia of Pharmaceutical Technology”, editedby Swarbrick, J. & J. C. Boylan, Marcel Dekker, Inc., New York, 1988ISBN 0-8247-2800-9.

[0093] A pharmaceutical composition comprising an active substanceserves as a drug delivery system. In the present context the term “drugdelivery system” denotes a pharmaceutical composition (a pharmaceuticalformulation or a dosage form) which upon administration presents theactive substance to the body of a human or an animal. Thus, the term“drug delivery system” embraces plain pharmaceutical compositions suchas, liquids, powders and sprays.

[0094] The choice of pharmaceutically acceptable excipients in acomposition for use according to the invention and the optimumconcentration thereof cannot generally be predicted and must bedetermined on the basis of an experimental determination thereof. Alsowhether a pharmaceutically acceptable excipient is suitable for use in apharmaceutical composition is generally dependent on which kind ofdosage form is chosen. However, a person skilled in the art ofpharmaceutical formulation can find guidance in e.g., “Remington: Thescience and practice of pharmacy” 20^(th) ed. Mack Publishing, EastonPa., 2000 ISBN 0-912734-04-3.

[0095] A pharmaceutically acceptable excipient is a substance, which issubstantially harmless to the individual to which the composition willbe administered. Such an excipient normally fulfils the requirementsgiven by the national drug agencies. Official pharmacopoeias such as theBritish Pharmacopoeia, the United States of America Pharmacopoeia andthe European Pharmacopoeia set standards for well-known pharmaceuticallyacceptable excipients.

[0096] In the following is given a review on relevant pharmaceuticalcompositions for use according to the invention. The review is based onthe particular route of administration. However, it is appreciated thatin those cases where a pharmaceutically acceptable excipient may beemployed in different dosage forms or compositions, the application of aparticular pharmaceutically acceptable excipient is not limited to aparticular dosage form or of a particular function of the excipient.

[0097] Parenteral Compositions:

[0098] For systemic application, the compositions according to theinvention may contain conventionally non-toxic pharmaceuticallyacceptable carriers and excipients according to the includingmicrospheres and liposomes.

[0099] The compositions for use according to the invention include allkinds of solid, semisolid and fluid compositions. Compositions ofparticular relevance are e.g. solutions, suspensions, emulsions.

[0100] The pharmaceutically acceptable excipients may include solvents,buffering agents, preservatives, chelating agents, antioxidants,stabilizers, emulsifying agents, suspending agents, diluents. Forexamples of the different agents see below.

[0101] In a most preferred embodiment, lyophilised powder of Emdogain®(BIORA AB, Malmö, Sweden) is dissolved in phosphate buffered saline(PBS) to a final concentration of 30 mg/ml.

[0102] Examples of other, equally preferred embodiments are lyophilisedpowder of Emdogain® (BIORA AB, Malmö, Sweden) dissolved in phosphatebuffered saline (PBS) to a final concentration of 0.01-50 mg/ml, such as0.01-10 mg/ml, 0.1-10 mg/ml, 0.01-5 mg/ml, 0.1-5 mg/ml, 0.01-1 mg/ml,0.1-1 mg/ml, or 0.01-0.05 mg/ml, comprising concentrations of about 0.01mg/ml, 0.02 mg/ml, 0.03 mg/ml, 0.04 mg/ml 0.05 mg/ml, 0.06 mg/ml, 0.07mg/ml, 0.08 mg/ml, 0.09 mg/ml, 0.1 mg/ml, 0.2 mg/ml, 0.3 mg/ml, 0.4mg/ml, 0.5 mg/ml, 1 mg/ml, 10 mg/ml, 20 mg/ml, 30 mg/ml, 35 mg/ml, 40mg/ml, 45 mg/ml, or 50 mg/ml.

[0103] Furthermore, as documented in example 2, the weight of the closeof enamel matrix derivatives administered to the mammal in need thereofis of course adjusted to the individual bodyweight of the mammal to betreated, and the desired effectiveness of EMD, and will most preferablybe in the range of about 0.01-100 mgEMD/kg bodyweight, such as about0.01-50 mgEMD/kg, 0.05-10 mg EMD/kg , 0.01-1 mg EMD/kg, 0.1-50 mgEMD/kg, 0.1-25 mg EMD/kg, 0.1-15 mg EMD/kg, or 0.1-10 mg EMD/kg, or0.1-1 mg EMD/kg bodyweight, depending further on whether enamel matrixderivatives is to be applied at a high or a low dosage.

[0104] A typical low dosage will thus include at most 0.01, 0.02, 0.03,0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.12, 0.15, 0.17, 0.2, 0.5, 1,1.2, 1.5, 1.6, 1.8, 2, 2.5, 5, 6, 7, 8, 9, or 9.9 mg EMD/kg bodyweight,whereas a typical high dosage will include at least 10, 15, 18, 20, 25,50, 75, or 100 mg EMD/kg bodyweight.

[0105] Dosages described above will be applied at least once/week, suchas at least 1, 2, 3, 4, 5, 6, 7, or more times/week for at least 1-30days, or alternatively for at least 10-90 days, or longer.

[0106] Examples of Various Agents:

[0107] Examples of solvents are but not limited to water, alcohols,blood, plasma, spinal fluid, ascites fluid and lymph fluid.

[0108] Examples of buffering agents are but not limited to citric acid,acetic acid, tartaric acid, lactic acid, hydrogenphosphoric acid,bicarbonates, phosphates, diethylamine etc.

[0109] Examples of chelating agents are but not limited to sodium EDTAand citric acid.

[0110] Examples of antioxidants are but not limited to butylated hydroxyanisole (BHA), ascorbic acid and derivatives thereof, tocopherol andderivatives thereof, cysteine, and mixtures thereof.

[0111] Examples of powder components are but not limited to alginate,collagen, lactose, powder which is able to form a gel when applied to awound (absorbs liquid/wound exudate).

[0112] Examples of diluents and disintegrating agents are but notlimited to lactose, saccharose, emdex, calcium phosphates, calciumcarbonate, calcium sulphate, mannitol, starches and microcrystallinecellulose.

[0113] Examples of binding agents are but not limited to saccharose,sorbitol, gum acacia, sodium alginate, gelatine, starches, cellulose,sodium coboxymethylcellulose, methylcellulose, hydroxypropylcellulose,polyvinylpyrrolidone and polyetyleneglycol.

LEGENDS TO FIGURES

[0114]FIG. 1:

[0115] Whole blood samples treated with LPS, PepG and matrix proteincompositions were incubated with 10 μl FITC or PE conjugated monoclonalantibodies. Expression of antigens were analysed by flow cytometry using10 μl anti-CD14, anti-CD44, anti-ICAM-1, as well as isotype negativecontrol anti-IgG₁ and anti-IgG₂. All samples are analysed on the FACScanusing the CellQuest software.

[0116]FIG. 2:

[0117] Amelogenins were extracted from immature pig enamel according toGestrelius et al. 1997. After solution in 30% acetonitril, the sampleswere separated on a Jasco HPLC using a size-exclusion column. The fourdistinct peaks represent the complex, the full length protein and thesoluble, processed amelogenin peptides.

[0118]FIG. 3:

[0119] Enamel matrix derivatives caused decreased production of thepro-inflammatory cytokine tumour necrosis factor alpha (TNF-α), whichwas paralleled by increased release of the anti-inflammatory cytokineIL-10.

LISTING OF REFERENCES

[0120] 1. Foster, S. J. 1992. Analysis of the autolysins of Bacillussubtilis 168 during vegetative growth and differentiation by usingrenaturing polyacrylamide gel electrophoresis. J Bacteriol. 174:464-70.

[0121] 2. Gestrelius, S., C. Andersson, D. Lidstrom, L. Hammarstrom, andM. Somerman. 1997. In vitro studies on periodontal ligament cells andenamel matrix derivative. J Clin Periodontol. 24:685-92.

[0122] 3. Hoang, A. M., T. W. Oates, and D. L. Cochran. 2000. In vitrowound healing responses to enamel matrix derivative [In ProcessCitation]. J Periodontol. 71:1270-7.

[0123] 4. Petinaki, E., S. Nikolopoulos, and E. Castanas. 1998. Lowstimulation of peripheral lymphocytes, following in vitro application ofEmdogain. J Clin Periodontol. 25:715-20.

[0124] Experimental Section

[0125] Experiment 1

[0126] The aim of the present study was to investigate potentialmodulatory effects of amelogenin on innate immune mechanisms in humanwhole blood. Of particular interest was the cytokine release andregulation of inflammatory surface receptors in response to bacterialcell wall products.

[0127] Materials and Reagents.

[0128] Peripheral venous blood was obtained from healthy staff membersand collected in vacutainers containing 0.129 M CitNa (Becton DickinsonVacutainer Systems Eur. Meylan Cedex, France). Lyophilised powder ofEmdogain® (BIORA AB, Malmö, Sweden) was dissolved in phosphate bufferedsaline (PBS) to a final concentration of 30 μg/ml. Escherichia coli026:B6 lipopolysaccharide (LPS; Difco Laboratories, Detroit, Mich., USA)was suspended in pyrogen free sterile saline and 10 ng/ml of LPS addeddirectly to the blood samples in microcentrifuge tubes. Lipoteichoicacid (LTA) was purchased from Sigma chemicals (St.Louis, Miss., USA).Peptidoglycan (PepG) was isolated from Staphylococcus aureus, aspreviously described (Foster, S. J. 1992 J Bacteriol. 174:464-70).Analysis of the autolysines of Bacillus subtilis 168 during vegetativegrowth and differentiation was performed by using renaturingpolyacrylamide gel electrophoresis, the autolysines were added directlyto the blood samples at a concentration of 10 μg/ml.

[0129] Whole Blood Experiments.

[0130] Peripheral venous blood was obtained from healthy staff membersand collected in vacutainers containing 0.129 M CitNa (Becton DickinsonVacutainer Systems Eur, Meylan Cedex, France). The whole blood sampleswere aliquoted into 1.5 ml microcentrifuge tubes (Sorenson, BioScienceInc., Salt Lake City, Utah, USA). Prior to stimulation with LPS, PepG orLTA, the blood samples were preincubated at 37C for 2 h with dissolvedEmdogain® at a final concentration of 0, 45, 150, 300, 450, 600, or 750g/ml.

[0131] The blood samples were then stimulated with 10 ng/ml of LPS, 10g/ml PepG or 100 g/ml LTA for 4 or 6 h, alternatively. The blood wassubsequently cooled on ice and centrifuged (14,000 g for 1 min), andplasma pipetted off for cytokine protein detection. As controls,equivalent volumes of 0.9% NaCl were added instead of the bacterialproducts. Basal cytokine levels were investigated immediately afterblood drainage.

[0132] Preliminary studies had shown that PepG and LTA give peak valuesof TNF-a, 6 h after stimulation, compared to 4 h with LPS stimulation.Accordingly, 4 h of LPS stimulation and 6 h of PepG and LTA stimulationwere chosen.

[0133] ELISA technique. Plasma concentrations of TNF-α, IL-6 and IL-10is determined with a commercially available solid-phase sandwich enzymelinked immunosorbent assay (ELISA) kit (PeliKine Compact, CLB Labs,Amsterdam, The Netherlands) according to the manufacturer'sinstructions. The detection limit of the TNF-α, IL-6 and IL-10 ELISA is3, 0.4 and 3 pg/ml, respectively. The plates are read at 450 nm in anELISA reader (Thermo max microplate reader, Molecular Devices, MenloPark, Calif., USA).

[0134] Antibody labelling and flow cytometry. Subsequent to stimulation,whole blood samples are immediately aliquoted in 100 μl into Falconpolystyrene tubes (Becton Dickinson, Lincoln Park, N.J., USA) andincubated with 10 μl FITC or PE conjugated monoclonal antibodies indarkness for 30 minutes at room temperature. Prior to washing andfixation, red blood cells are lysed using FACS Lysing Solution (BectonDickinson, San Jose, Calif., USA). The samples are washed three timeswith CellWash (Becton Dickinson) before fixation with formaldehyde 1%(CellFIX, Becton Dickinson, Erembodegem, Belgium). Expression ofantigens are analysed by flow cytometry using 10 μl anti-CD14 (18D11,Diatec AS, Oslo, Norway), anti-CD44 (Diatec AS, Oslo, Norway),anti-ICAM-1 (Diatec AS, Oslo, Norway), as well as isotype negativecontrol anti-IgG₁ (1B9) and anti-IgG₂ (5A7) (both Diatec AS). Allsamples are analysed on the FACScan (Becton Dickinson) using theCellQuest software. The results are represented in FIG. 1.

[0135] In each experiment, the monocyte population is identified as CD14positive cells with characteristic location in the scatter diagram.Fluorescence intensity (FI) is measured in the monocyte population afterappropriate gating according to these features, and mean FI (geometricmean) is recorded for each measurement.

[0136] Statistical Analyses. Data is presented as means±standard errorof the mean (SEM). The data are analysed by 1-way analysis of variance(ANOVA) followed by the Tukey test. P<0.05 is considered significant.

[0137] As seen in FIG. 3, enamel matrix derivatives caused decreasedproduction of the proinflammatory cytokine tumour necrosis factor alpha(TNF-α), which was paralleled by increased release of theanti-inflammatory cytokine IL-10. The results indicate that enamelmatrix derivatives have potent immunomodulatory properties. It is alsotempting to suggest that enamel matrix derivatives may have therapeuticpotentials in sepsis with multiple organ failure. In addition, enamelmatrix derivatives may have a therapeutic potential in other diseasesassociated with acute or chronic inflammation, such as inflammatorybowel disease, rejection of transplanted organs or orthopaedic implants,rheumatoid arthritis, arteriosclerosis and several others.

[0138] Experiment 2

[0139] In this study using a pig model of sepsis, the therapeuticpotential of enamel matrix derivatives was examined in vivo. The modelis based on continuous infusion of E. Coli LPS, giving rise to an acuteinflammatory response. After a while (typically after half to one hourof LPS injection) the animal enters into septic shock mirroringimportant patho-physiological features of severe sepsis. The model is apowerful tool linking in vitro findings with possibly clinical trials.

[0140] Enamel matrix derivatives (max dosage5 mg/kilo bodyweight, i.e. acalculated serum concentration of about 100 mg/l) were administered byan i.v. bolus injection to the pigs 30 minutes before onset of sepsis(=injection of LPS, 1.7 μg/kilo bodyweight/hour). The half life forenamel matrix derivatives in blood is reported to be 240 minutes. Thus,to maintain a therapeutic serum level of EMD, an i.v. transfusion set upwas used to deliver 50 mg enamel matrix derivatives in 100 ml Ringer'ssolution (pH=6) per hour. For control, three “sham” pigs are treatedidentically, except for administration of EMD, which was substitutedwith injection and transfusion with serum albumin (“placebo”).

[0141] Haemodynamic parameters were registered continuously, with hourlyrecordings until end of experiment at 5 h after infusion of bacterialtoxins (LPS). Blood samples and recording of clinical parameters weretaken before the experiment's start (time zero), immediately after startof enamel matrix derivatives or placebo injection (time zero-a),immediately after start of LPS injection (time one) and every hour afteronset of sepsis from the hepatic and portal vein, the abdominal artery,and the pulmonary artery, and analysed for blood homeostasis parameters,organ function markers (serum aspertate aminotransferase (ASAT), serumalanine aminotransferase (ALAT), and serum alkaline phosphatase activityas markers for liver function, and gamma-glutamyltransferase (γ-GT) andCreatinine as marker for kidney function) and inflammatory mediators(TNF-α, IL-1β and IL-6). The organ function markers were analysed inblood sampled from the pig hourly during the experiment at predefinedtime points. The blood samples were analysed using standard hospitalprocedures in the haematological laboratory of the Norwegian NationalHospital. The inflammatory markers were analysed in the same bloodsamples by the use of ELISA, applying the Porcine TNF-α/TNFSF2Quantikine P ELISA kit (#PTA00), the Porcine IL-6 Quantikine Pimmunoassay kit (#P6000), and the Porcine IL-1β/IL-1F2 Quantikine PELISA kit (#PTA00), all from R&D Systems Inc. (Minneapolis, Minn., USA).The standard procedures from the manufacturer were used withoutomissions or adaptations.

[0142] After the experiment was completed (death of the animal, or after5 hours with continuous injections of enamel matrix derivatives orplacebo and LPS), the animals were euthanised and necropsy performed.Internal circulatory organs were examined macroscopically and tissueswere sampled for histological evaluation according to the research plan.

[0143] Results

[0144] PIG 1

[0145] Physical data: Female; 23.5 kg total bodyweight; Healthy

[0146] Surgery: No complications

[0147] Treatment: Placebo (25 mg serum albumin in 50 ml PBS bolusinjection, 10 mg SA. in 100 ml Ringer's solution per hour transfusion,total SA dosage=75 mg), LPS (40 μg/hour in 4.5 ml PBS, continuousinjection for 5 hours; total LPS dosage=200 μg).

[0148] Haemodynamic observations: The animal showed no response toplacebo injection. A strong initial response to LPS injection wasobserved followed by a septic shock condition that lasted throughout theexperiment. The onset of shock was before one hour after initiation ofLPS injection.

[0149] Anatomy and macroscopic appearance of circulatory organs atdeath: All circulatory organs had normal anatomy. Signs of pathology wasobserved in the liver which showed signs of congestion and haemostasis.

[0150] Haematology: The animal initially showed normal haematologicalvalues. Most values stayed within the normal range throughout theexperiment. The only exception was observed at four and five hours afterLPS injection, when an increase in B-neutrophiles and a decrease in Blymphocytes was observed.

[0151] Biochemistry: All enzyme activities and Creatinine stayed withinnormal values throughout the experiment. A slow but steady decrease increatinine was observed, but the values never decreased below the norm.The pig initially showed a slight increase in ALP, but this normalizedbefore LPS was administered. ASAT values were also on the high sidethroughout the experiment, but did not show any significant change fromLPS injection.

[0152] Cytokines: The pig showed a rapid increase in TNF values afteradministration of LPS. The value peaked above detection limit (1500μg/l) before one hour after start of LPS injection and stayed at thislevel throughout the experiment. Also IL-6 increased rapidly followingthe onset of LPS injection. The IL-6 level increased steadily all theway through the experiment ending at a level of 3500 g/l. IL-1β showed aslow increase caused by the LPS injection, ending at about 700 μg/lafter 5 hours.

[0153] Comments: Haemodynamic parameters indicated hepatic and lungfailure towards the end of the experiment. Low and declining diuresisindicated renal insufficiency. First signs of organ insufficiency wasobserved at two hours after LPs injection started. Tremor was observedfrom 30 minutes after LPS administration. Haematology and biochemistryvalues indicated that the life support systems were operated correctly,and that the observed effects were caused by the toxin injection and notby trauma from surgery. The Inflammatory status indicated that the pigwas suffering from a septic shock. There were no signs of recovery inthe cytokine values. It is very unlikely that this pig could havesurvived the experiment.

[0154] PIG 2

[0155] Physical data: Female; 21.5 kg total bodyweight; Healthy

[0156] Surgery: No complications

[0157] Treatment: Enamel matrix derivatives (21.5 mg enamel matrixderivatives in 50 ml PBS bolus injection, 10 mg enamel matrixderivatives in 100 ml Ringer's solution per hour transfusion, totalenamel matrix derivatives dosage=71.5 mg), LPS (37 μg/hour in 4.5 mlPBS, continuous injection for 5 hours; total LPS dosage=185 μg).

[0158] Haemodynamic observations: The animal showed no response to thebolus injection of EMD. A very rapid and strong initial response to LPSinjection was observed (tachycardia and increased arterial flow)followed by an onset of a septic shock condition at about one hour afterstart of LPS injection. At this stage, early signs or multi-organfailure was observed. However, at later time points the haemodynamicvalues stabilized and organ functions recovered.

[0159] Anatomy and macroscopic appearance of circulatory organs atdeath: All circulatory organs had normal anatomy. Macroscopic signs ofpathology were not observed in organs examined.

[0160] Haematology: The animal initially showed normal haematologicalvalues. All values stayed within the normal range throughout theexperiment.

[0161] Biochemistry: All enzyme activities and creatinine stayed withinnormal values throughout the experiment. The pig initially showed aslight increase in ALP, but this stayed unchanged by enamel matrixderivatives or LPS injections. ASAT values increased slightly towardsthe very end of the experiment, but did not show any significant changefrom enamel matrix derivatives or LPS injection.

[0162] Cytokines: Enamel matrix derivatives injection did not provokeany increase in the analysed circulating cytokines. However, the pigshowed a rapid increase in TNF values after administration of LPS. Thevalue peaked above detection limit (1500 μg/l) before one hour afterstart of LPS injection and stayed at this level for about four hours.After four hours, the TNF value decreased and showed sign ofnormalizing. Also IL-6 increased rapidly following the onset of LPSinjection. The IL-6 level increased steadily through the experimentpeaking at 4800 μg/l after four hours. After five hours the IL-6 valuesseemed to decrease, but because of the (planned) termination of theexperiment, this trend was not confirmed. IL-1β showed a slow increasecaused by the LPS injection, ending at about 1000 μg/l after 5 hours.

[0163] Comments: The animal seemed to recover from an obvious septicshock and onset of organ failure, as observed both by haemodynamicparameters and by recovery of renal function (diuresis) and reduction inascites. Tremor occurred from one hour after LPS administration. Thesefindings are uncommon, especially following such a strong initialresponse to endotoxin (LPS). Haematology and biochemistry valuesindicated that the life support systems were operated correctly, andthat the observed effects were caused by the toxin injection and not bytrauma from surgery. The Inflammatory status indicated that the piginitially was suffering from a septic shock. There were, however, signsof slow recovery in the IL-6 and TNF values. IL-1 values seemedunaffected by enamel matrix derivatives administration. Given time and acontinuing decrease in the inflammatory cytokine levels, this pig wouldmost likely have survived the experiment.

[0164] PIG 3

[0165] Physical data: Female; 25.0 kg total bodyweight; Healthy

[0166] Surgery: No complications

[0167] Treatment: Placebo (125 mg SA. in 100 ml PBS bolus injection, 50mg SA. in 100 ml Ringer's solution per hour transfusion, total SAdosage=375 mg), LPS (43 μg/hour in 4.5 ml PBS, continuous injection for5 hours; total LPS dosage=215 μg).

[0168] Haemodynamic observations: The animal showed no response to theplacebo injection. A low to medium initial response to LPS injection wasobserved followed by a septic shock condition that lasted throughout theexperiment. The onset of shock was about one hour after initiation ofLPS injection.

[0169] Anatomy and macroscopic appearance of circulatory organs atdeath: All circulatory organs had normal anatomy. Signs of pathologywere only observed in the liver which showed signs of congestion(swelling and redness) and haemostasis.

[0170] Haematology: The animal initially showed normal haematologicalvalues. Most values stayed within the normal range throughout theexperiment. The only exception was observed at four and five hours afterLPS injection, when an increase in B-neutrophiles and a decrease in Blymphocytes was observed.

[0171] Biochemistry: Most enzyme activities stayed within normal valuesthroughout the experiment. A slow but steady decrease in creatinine wasobserved, and after two hours after LPS injection, the creatinine leveldecreased below the normal range and stayed low for the rest of theexperiment. The pig initially showed an increased ALP activity, but thisvalue was stable and unaffected by the LPS injection. The Inflammatorystatus indicated that the pig was suffering from a septic shock. Therewere no signs of recovery in the cytokine values. It is very unlikelythat this pig could have survived the experiment.

[0172] Cytokines: The pig showed a rapid increase in TNF values afteradministration of LPS. The value peaked above detection limit (1500μg/l) before one hour after start of LPS injection and stayed at thislevel throughout the experiment. Also IL-6 increased rapidly followingthe onset of LPS injection. The IL-6 level increased steadily all theway through the experiment peaking at 5000 μg/l at three hours after LPSinjection, ending at a level of 3900 μg/l. IL-1β showed a slow increasecaused by the LPS injection, ending at about 700 μg/l after 5 hours.

[0173] Comments: Haemodynamic parameters indicated hepatic failuretowards the end of the experiment. Low diuresis indicate renalinsufficiency. Signs of organ failure were observed from three hours onafter LPS injection started. Tremor was evident 30 minutes after LPSadministration. Haematology and biochemistry values indicated that thelife support systems were operated correctly, and that the observedeffects were caused by the toxin injection and not by trauma fromsurgery. The Inflammatory status indicated that the pig was sufferingfrom a septic shock. There were no signs of recovery in the cytokinevalues. It is very unlikely that this pig could have survived theexperiment.

[0174] PIG 4

[0175] Physical data: Female; 24.5 kg total bodyweight; Healthy

[0176] Surgery: No complications

[0177] Treatment: Enamel matrix derivatives (125 mg enamel matrixderivatives in 100 ml PBS bolus injection, 50 mg enamel matrixderivatives in 100 ml Ringer's solution per hour transfusion, totalenamel matrix derivatives dosage=375 mg), LPS (42 μg/hour in 4.5 ml PBS,continuous injection for 5 hours; total LPS dosage=210 μg).

[0178] Haemodynamic observations: The animal showed no response to thebolus injection of EMD. A strong initial response to LPS injection wasobserved followed by a weak septic shock condition at about three hoursafter start of LPS injection. At this stage early signs or lung failurewere observed. However, at later time points, the haemodynamic valuesstabilized and all organ functions recovered.

[0179] Anatomy and macroscopic appearance of circulatory organs atdeath: All circulatory organs had normal anatomy. Macroscopic signs ofpathology were not observed in organs examined.

[0180] Haematology: The animal initially showed normal haematologicalvalues. All values stayed within the normal range throughout theexperiment.

[0181] Biochemistry: All enzyme activities and creatinine stayed withinnormal values throughout the experiment. The pig initially showed aslightly increased ALP activity, but this stayed unchanged by enamelmatrix derivatives or LPS injections. ASAT values increased slightlytowards the very end of the experiment, but did not show any significantchange directly associated with the enamel matrix derivatives or LPSinjection.

[0182] Cytokines: Enamel matrix derivatives injection did not provokeany increase in the analysed circulating cytokines. However, the pigshowed a rapid increase in TNF values after administration of LPS. Thevalue peaked above detection limit (1500 μg/l) before one hour afterstart of LPS injection and stayed at this level throughout theexperiment. Also IL-6 increased rapidly following the onset of LPSinjection. The IL-6 level increased steadily through the experimentrising above detection level (>5000 μg/l) after three hours. At fourhours the IL-6 values seemed to decrease, and this trend was confirmedalso at five hours. IL-1β showed a slow, steady increase caused by theLPS injection, ending at about 700 μg/l after 5 hours.

[0183] Comments: The animal seemed to be protected from severe septicshock and organ failure, as observed both by clinical and haemodynamicparameters. No organ failure was observed, but diuresis was lowthroughout the experiment. There was only minute production of ascites,and no tremor was observed. These findings are uncommon, especiallyfollowing a strong initial response to endotoxin (LPS). Haematology andbiochemistry values indicated that the life support systems wereoperated correctly, and that the observed effects were caused by thetoxin injection and not by trauma from surgery. The Inflammatory statusindicate that the pig initially suffered from onset of a septic shock.There were, however, signs of slow recovery in the IL-6 and TNF values.IL-1β values seemed unaffected by enamel matrix derivativesadministration. Given time and a continuing decrease in the inflammatorycytokine levels, and the good haemodynamic condition, it is believedthat this pig would have survived the experiment.

[0184] PIG 5

[0185] Physical data: Female; 25.0 kg total bodyweight; Healthy

[0186] Surgery: No complications

[0187] Treatment: Enamel matrix derivatives (125 mg enamel matrixderivatives in 100 ml PBS bolus injection, 50 mg enamel matrixderivatives in 100 ml Ringer's solution per hour transfusion, totalenamel matrix derivatives dosage=375 mg), LPS (43 μg/hour in 4.5 ml PBS,continuous injection for 5 hours; total LPS dosage=215 μg).

[0188] Haemodynamic observations: The animal showed an initial increaseof lung blood pressure as response to the very start of bolus injectionof EMD. The effect was very transient (minutes) and not dosagedependent, indicating that the enamel matrix derivatives solution had abrief vasoconstrictive effect on the lung capillaries. The observedeffect was normalized before the bolus injection was completed. No othereffects was observed from the enamel matrix derivatives injection. Thisanimal did not show any profound response to LPS injection at all. Nosigns of a septic shock condition was observed and organ circulation wasstable throughout the experiment. The LPS flow and the position of theLPS catheter was controlled several times during the experiment toensure that LPS was delivered according to the experimental plan.

[0189] Anatomy and macroscopic appearance of circulatory organs atdeath: All circulatory organs had normal anatomy. Macroscopic signs ofpathology was not observed in organs examined.

[0190] Haematology: The animal initially showed normal haematologicalvalues, except for the count of white blood cells that was significantlyincreased (twice the normal value) from the start of surgery (0-sample).The relative levels of the different white blood cells appeared normalhowever, and remained stable during the experiment. All other valuesstayed within the normal range throughout the experiment.

[0191] Biochemistry: All enzyme activities and creatinine stayed withinnormal values throughout the experiment, except for ALP, which level wasincreased from the start of the experiment, but remained stablethroughout the observation period. The ALP level remained unchanged byenamel matrix derivatives or LPS injections. ASAT values increasedslightly from LPS injection to the end of the experiment. This increasewas however very small and peaked barely above the normality limit.

[0192] Cytokines: Enamel matrix derivatives injection did not provokeany increase in the analysed circulating cytokines. However, this pigshowed a significantly increased TNF value even before the experimentstarted (>1500 μg/l). This value stayed above detection range until 2hours after administration of LPS. However, from then on a rapiddecrease in TNF values was observed, and the TNF level was nearlynormalized before the end of the experiment. IL-6 increased rapidlyfollowing the onset of LPS injection. The IL-6 level increased steadilyuntil two hours after LPS injection peaking at 2500 μg/l. The IL-6levels then tapered off, reaching normal level at five hours after LPSinjection. IL-1β showed a slow, steady increase caused by the LPSinjection, ending at about 900 μg/l after 5 hours.

[0193] Comments: The animal seemed to be completely protected from LPSinduced septic shock and organ failure, as observed both by clinical andhaemodynamic parameters. No organ failure was observed, and diuresis wasgood and stable throughout the experiment. There was only minuteproduction of ascites, and no tremor was observed. It is extremelyuncommon that animals fail to respond to central endotoxin (LPS)injections in this way. Haematology and biochemistry values indicatedthat the life support systems were operated correctly, and that theobserved effects were caused by the toxin injection and not by traumafrom surgery. The Inflammatory status indicate that the pig initiallysuffered from a weak onset of septic shock. There was however, clearsigns of rapid recovery in the IL-6 and TNF values. IL-1β values seemedunaffected by enamel matrix derivatives administration. There is littledoubt that this animal, if allowed, would have survived the experiment.

[0194] PIG 6

[0195] Physical data: Female; 23.0 kg total bodyweight; Healthy

[0196] Surgery: No complications

[0197] Treatment: Enamel matrix derivatives (115 mg enamel matrixderivatives in 100 ml PBS bolus injection, 50 mg enamel matrixderivatives in 100 ml Ringer's solution per hour transfusion, totalenamel matrix derivatives dosage=365 mg), LPS (39 μg/hour in 4.5 ml PBS,continuous injection for 5 hours; total LPS dosage=195 μg).

[0198] Haemodynamic observations: The animal showed an initial increaseof lung blood pressure as response to the bolus injection of EMD. Theresponse was spontaneous and transient (minutes) and not dosagedependent, indicating that enamel matrix derivatives injection had abrief vasoconstrictive effect on the lung capillaries. The effect wasnormalized before the bolus injection was completed.

[0199] A strong initial response to LPS injection was observed (rapid,transient increase in heart rate and arterial flow), followed by amoderately hyperbar condition that lasted throughout the experiment. Nosigns of septic shock was observed, nor were there any signs of organfailure.

[0200] Anatomy and macroscopic appearance of circulatory organs atdeath: All circulatory organs had normal anatomy. Macroscopic signs ofpathology was not observed in organs examined.

[0201] Haematology: The animal initially showed normal haematologicalvalues. All values stayed within the normal range throughout theexperiment.

[0202] Biochemistry: All enzyme activities and creatinine stayed withinnormal values throughout the experiment, except for ALP and ASAT thatwere slightly increased from the start of the experiment, but remainedstable throughout the observation period. The ALP and ASAT levelsremained unchanged by enamel matrix derivatives or LPS injections.

[0203] Cytokines: Enamel matrix derivatives injection did not provokeany increase in the analysed circulating cytokines. However, the pigshowed a rapid increase in TNF values after administration of LPS. Thevalue peaked above detection limit (1500 μg/l) before one hour afterstart of LPS injection and stayed at this level for about three hours.After three hours, the TNF value decreased rapidly, and normalized atthe end of the observation period. Also IL-6 increased rapidly followingthe onset of LPS injection. The IL-6 level peaked at 2700 μg/l, twohours after LPS injection. However, at the end of the experiment theIL-6 value had decreased to nearly normal value (350 μg/l). IL-1β showeda slow, steady increase caused by the LPS injection, peaking at about1200 μg/l after 4 hours.

[0204] Comments: This animal seemed to be protected from severe septicshock and organ failure, as observed both by haemodynamic and clinicalparameters. No organ failure was observed and diuresis was high andstable throughout the experiment. There was only minute production ofascites, and tremor was observed only after about four hours of LPSinfusion. These findings are uncommon, especially following a stronginitial response to endotoxin (LPS), as observed here. Haematology andbiochemistry values indicated that the life support systems wereoperated correctly, and that the observed effects were caused by thetoxin injection and not by trauma from surgery. The inflammatory statusindicated that the pig initially suffered from a of strong septic shock(rapid, strong increase in TNF and IL-6). There were, however, clearsigns of total remission in the IL-6 and TNF values. IL-1β values seemedunaffected by enamel matrix derivatives administration. There is littledoubt that this animal, if allowed, would have survived the experiment.

SUMMARY

[0205] Controls (pig 1 and 3)

[0206] The controls both behaved normally, i.e. they both entered into aseptic shock situation after about 30 minutes after the onset of LPSinjection. Both pigs survived 5 hours of LPS injection, however, theyshowed clear clinical signs of severe, uncontrolled septic shock withincreased temperature, multi-organ failure, renal insufficiency,hypotension and reduced cardiac output. Also analysis of the bloodshowed clear signs of septic shock. The inflammatory status also matchedan acute toxin induced septic shock with strong expression of both TNF-αand Interleukin-6. The condition of these pigs towards the end of theexperiment was so severe that it was judged to be incompatible withcontinued living.

[0207] Low Enamel Matrix Derivatives Dosage (pig 2)

[0208] The pig administered the low enamel matrix derivatives dosagealso entered into a septic shock situation. However, the onset of theacute inflammatory response to LPS was delayed with about 30-60 minuteswhen compared to the sham animals. After the onset of sepsis, the pigstabilized and then slowly recovered from the shock. This animal showedslightly better clinical parameters and did not show signs of renalfailure (as measured by diuresis). The pig was euthanised as planned 5hours after onset of LPS injection. The bolus injection and thefollowing transfusion with a “low” dosage of enamel matrix derivativesdid not provoke any clinical signs of adverse reactions in this pig, nordid any monitored parameter respond to the enamel matrix derivativesinjection. This pig also showed a decreasing trend in TNF and IL-6values. This observation, together with the clinical observationssuggest that the animal was recovering from the shock.

[0209] High Enamel Matrix Derivatives Dosage (pig 4, pig 5 and pig 6)

[0210] None of the three animals administered high enamel matrixderivatives dosages did experience a severe septic shock situation fromthe LPS injection. Two animals (pig 4 and pig 6) experienced an initial,but transient endotoxin response, but the situation quickly stabilizedand the animals quickly recovered and remained “controlled” throughoutthe experiment. The third animal (pig 5) did not respond to the LPSinjection at all. None of these animals showed signs of multi-organfailure or renal insufficiency. Only one of the pigs (pig 6) experiencedthe typical tremor induced by LPS injection. The onset of tremor wassignificantly delayed (4 hours), as compared to the controls. The pigswere euthanised as planned 5 hours after the initiation of LPSinjection.

[0211] The bolus injections and the following transfusion with “high”dosages of enamel matrix derivatives did not provoke any persistentclinical signs or adverse reactions in these pigs. In two of the pigs(pig 5 and pig 6), an initial response to the bolus injection wasobserved. This response was observed as a rapid, but transient increasein pulmonary arterial pressure indicating that the central bolusinjection of the high concentration enamel matrix derivatives solution(ca. 1.25 mg/ml) induced a spontaneous constriction of pulmonaryarterioles and/or capillaries. The effect was immediate, and was notdosage related. The observed effect normalized within minutes and thepigs were stable, showing normal haemodynamic parameters and clinicalstatus before the completion of the bolus injection.

[0212] These pigs also showed a decreasing trend in TNF and IL-6 valuesafter the initial LPS response. In fact, in two of the three animals(pig 5 and pig 6) the TNF and IL-6 values nearly normalized before theend of the observation period. This observation, together with theclinical observations, suggest that these animals were protected fromthe LPS induced shock and subsequent multi organ failure. The enamelmatrix derivatives injection administered in these pigs protected theanimals from severe damage from the toxin injection, and thus rescuedthe animals from experiencing a septic shock. All three animals wouldhave survived the experiment.

1. Use of an active enamel substance and/or a preparation of an activeenamel substance for the manufacture of a pharmaceutical composition forthe prevention and/or treatment of a condition in a mammal, saidcondition being characterised by an imbalance in the immune response ofsaid mammal to an immunogen.
 2. Use according to claim 1, wherein saidactive enamel substance modulates a function of the immune mechanism ofsaid mammal.
 3. Use according to claim 1 or 2, wherein at least a partof said immune system reacts hypersensitively to said immunogen.
 4. Useaccording to any of claims 1-3, wherein at least a part of said immunesystem is stimulated non-discriminatingly.
 5. Use according to any ofclaims 1-4, wherein at least a part of said immune system fails to reactto said stimuli.
 6. Use according to any of claims 1-5, wherein theimbalance of said immune response is local.
 7. Use according to any ofclaims 1-6, wherein the imbalance of said immune response is systemic.8. Use according to any of claims 1-7, wherein the condition ischaracterised as an autoimmune disease.
 9. Use according to claim 8,wherein said auto-immune disease is selected from the group consistingof rheumatoid arthritis, diseases of the skin, allergy, sclerosis,arteriosclerosis, multiple sclerosis, asthma, psoriasis, lupus, diabetesmellitus, myasthenia gravis, chronic fatigue syndrome, fibromyalgia,Crohn's disease, Hashimoto's thyroiditis, Grave's disease, Addison'sdisease, Guillian Barre syndrome and scleroderma.
 10. Use according toclaim 7, wherein said condition is characterised as a systemicinflammation and/or infection.
 11. Use according to claim 10, whereinsaid systemic condition is associated with trauma, burns, surgery,dialysis, shock and/or bacterial or viral infection.
 12. Use accordingto claim 10, wherein said systemic condition is associated with adisease selected from the group consisting of extracorporeal membraneoxygenation (ECMO), shock, systemic inflammatory response syndrome(SIRS), sepsis, multi organ failure, rejection of an implant such asorgan and/or prosthesis, heart and lung bypass surgery, chronicinflammation, asthma and/or stress related disease in the lung,bacterial infection, gangrene, soft tissue infection, and woundinfection.
 13. Use according to claim 11 or 12, wherein said infectionis caused by a contamination with a bacterium selected from the groupconsisting of gram negative microbes and gram positive microbes.
 14. Useaccording to claim 13, wherein said infection is caused by an endo-toxinand/or exotoxin produced by said microbe.
 15. Use according to claim 14,wherein said infection is caused by a PepG from Staphylococcus aureus.16. Use according to claim 14, wherein said infection is caused by anLPS from Escherichia coli.
 17. Use according to any of claims 11-16,wherein said active enamel substance and/or said preparation comprisedin said pharmaceutical composition inactivates the toxic effect of theendo-toxin and/or exo-toxin produced by said microbe by binding to saidendo-toxin and/or exo-toxin produced by said microbe.
 18. Use accordingto any of claims 11-16, wherein said active enamel substance and/or saidpreparation comprised in said pharmaceutical composition inactivates thetoxic effect of the endo-toxin and/or exo-toxin produced by said microbeby binding to the cellular receptor for said endo-toxin and/or exo-toxinproduced by said microbe.
 19. Use according to any of the precedingclaims, wherein said active enamel substance and/or said preparationcomprised in said pharmaceutical composition modulates the conception ofT-helper cell activation in said mammal.
 20. Use according to any of thepreceding claims, wherein said active enamel substance and/or saidpreparation comprised in said pharmaceutical composition modulatesinter- and/or intra-cellular signalling substances.
 21. Use according toclaim 20, wherein said active enamel substance and/or said preparationcomprised in said pharmaceutical composition modulates the level ofexpression, release and/or function of at least one cytokine involved inthe immune mechanism of said mammal.
 22. Use according to any of thepreceding claims, wherein said active enamel substance and/or saidpreparation comprised in said pharmaceutical composition modulates acellular signalling cascade involved in said immune mechanism of saidmammal.
 23. Use according to claim 22, wherein said active enamelsubstance and/or said preparation comprised in said pharmaceuticalcomposition interacts with a cell surface receptor and/or a cell surfacestructure, selected from the group consisting of any ICAM, HLA antigen,Interleukin receptor, PDGF receptor, TGF receptor, TNF receptor, EGFreceptor, CD44 or Integrine.
 24. Use according to any of the precedingclaims, wherein said pharmaceutical composition is administeredsystemically.
 25. Use according to any of the preceding claims, whereinsaid active enamel substance is an enamel matrix, enamel matrixderivative and/or enamel matrix protein.
 26. Use according to any of thepreceding claims, wherein said active enamel substance is selected fromthe group consisting of enameling, amelogenins, non-amelogenins,proline-rich non-amelogenins, tuftelins, derivatives thereof andmixtures thereof.
 27. Use according to any of the preceding claims,wherein said active enamel substance is selected from the groupconsisting of amelogenins proteins and/or peptides, derivatives thereofand mixtures thereof.
 28. Use according to any of the preceding claims,wherein said active enamel substance has a molecular weight of at themost about 120 kDa such as, at the most 100 kDa, 90 kDa, 80 kDa, 70 kDaor 60 kDa as determined by SDS PAGE electrophoresis.
 29. Use accordingto any of the preceding claims, wherein said active enamel substanceand/or said preparation comprises an amelogenin processing product of nomore than 13 kDa.
 30. Use according to claim 29, wherein said processedamelogenin product is selected from a group consisting of solubleamelogenin based peptides with an amino acid length betweenapproximately 2-13 kDa, including a soluble amelogenin based peptidewith an amino acid length between 2-4.5 kDa, 3-5.5 kDa, 4-6.5 kDa, 5-7.5kDa, or 5-13 kDa.
 31. Use according to any of the preceding claims,wherein said processed amelogenin product is comprised in the eluaterepresented by the third and/or fourth peak of an HPLC analysis ofdegraded amelogenin as shown in FIG.
 2. 32. Use according to any of thepreceding claims, wherein said processed amelogenin product is a 5 kDapolypeptide.
 33. Use according to any of the preceding claims, whereinsaid active enamel substance has a molecular weight of up to about40,000.
 34. Use according to any of the preceding claims, wherein saidactive enamel substance has a molecular weight of between about 5,000and about 25,000.
 35. Use according to any of the preceding claims,wherein the major part of said active enamel substance has a molecularweight of about 20 kDa.
 36. Use according to any of the precedingclaims, wherein said preparation of an active enamel substance containsa mixture of active enamel substances with different molecular weights.37. Use according to any of the preceding claims, wherein saidpreparation of an active enamel substance comprises at least twosubstances selected from the group consisting of amelogenins,proline-rich non-amelogenins, tuftelin, tuft proteins, serum proteins,salivary proteins, ameloblastin, sheathlin, and derivatives thereof. 38.Use according to any of the preceding claims, wherein the proteincontent of said active enamel substance in the preparation is in a rangeof from about 0.005% w/w to 100% w/w such as, about 5-99% w/w, about10-95% w/w, about 15-90% w/w, about 20-90% w/w, about 30-90% w/w, about40-85% w/w, about 50-80% w/w, about 60-70% w/w, about 70-90% w/w, orabout 80-90% w/w.
 39. Use according to any of the preceding claims,wherein the pharmaceutical composition further comprises apharmaceutically acceptable excipient.
 40. Use according to claim 39,wherein the pharmaceutically acceptable excipient is phosphate buffersaline (PBS).
 41. A method for treating and/or preventing a condition ina mammal in need thereof by administering an effective amount of apharmaceutical composition comprising an active enamel substance and/ora preparation of an active enamel substance, said condition beingcharacterised by an imbalance in the immune response of said mammal dueto external and/or internal stimuli.
 42. A method according to claim 41,wherein active enamel substance modulates a function of the immunemechanism in a mammal in need thereof.
 43. A method according to claim41 or 42, wherein the imbalance of the immune response is ahypersensitive reaction to said stimuli.
 44. A method according to anyof claims 41-43, wherein the imbalance of the immune response ischaracterised by a failure to react to said stimuli.
 45. A methodaccording to any of claims 41-43, wherein the imbalance of the immuneresponse is characterised by a non-discriminating stimulation of saidimmune system.
 46. A method according to any of claims 41-45, whereinthe imbalance of the immune response of said mammal is local.
 47. Amethod according to any of claims 41-46, wherein the imbalance of theimmune response is systemic.
 48. A method according to claim 47, whereinthe condition is characterised as a systemic inflammation and/orinfection.
 49. A method according to claim 47 or 48, wherein thesystemic condition is associated with trauma, burns, surgery, dialysis,shock and/or bacterial or viral infection.
 50. A method according to anyof claims 47-49, wherein the systemic condition is associated withextracorporeal membrane oxygenation (ECMO), shock, systemic inflammatoryresponse syndrome (SIRS), sepsis, multi organ failure, rejection of animplant such as organ and/or prosthesis, heart and lung bypass surgery,chronic inflammation, asthma and stress related disease in the lung,bacterial infection, gangrene, soft tissue infection, and/or woundinfection.
 51. A method according to any of claims 47-50, wherein theinfection is caused by contamination with a selected from the groupconsisting of gram negative microbes and gram positive microbes.
 52. Amethod according to any of claims 47-51, wherein the infection is causedby an endotoxin and/or exo-toxin produced by said microbe.
 53. A methodaccording to claim 41-46, wherein the imbalance of the immune responseis an autoimmune disease.
 54. A method according to claim 53, whereinthe auto-immune disease is selected from but not limited to rheumatoidarthritis, diseases of the skin, allergy, sclerosis, arteriosclerosis,multiple sclerosis, asthma, psoriasis, lupus, diabetes mellitus,myasthenia gravis, chronic fatigue syndrome, fibromyalgia, Crohn'sdisease, Hashimoto's thyroiditis, Grave's disease, Addison's disease,Guillian Barre syndrome and scleroderma.
 55. A method according to anyof claims 41-54, wherein the active enamel substance in thepharmaceutical composition modulates the conception of T-helper cellactivation in said mammal.
 56. A method according to any of claims41-55, wherein the active enamel substance in the pharmaceuticalcomposition modulates intercellular signalling substances.
 57. A methodaccording to claim 56, wherein the active enamel substance in thepharmaceutical composition modulates the level of expression, releaseand/or function of cytokines involved in the immune mechanism of saidmammal.
 58. A method according to claim 41-57, wherein the active enamelsubstance in the pharmaceutical composition modulates a cellularsignalling cascade involved in the immune mechanism of said mammal. 59.A method according to claim 58, wherein the active enamel substancecomprised in the pharmaceutical composition interacts with a cellsurface receptor and/or a cell surface structure, selected from thegroup consisting of any ICAM, HLA antigen, Interleukin receptor, PDGFreceptor, TGF receptor, TNF-receptor, EGF receptor, CD44 or Integrine.60. A method according to any of the claims 41-58, wherein thepharmaceutical composition comprising an active enamel substance isadministered systemically.
 61. A method according to any of the claims41-60, wherein the active enamel substance is enamel matrix, enamelmatrix derivatives and/or enamel matrix proteins.
 62. A method accordingto any of the claims 41-61, wherein the active enamel substance isselected from the group consisting of enameling, amelogenins,non-amelogenins, proline-rich non-amelogenins, tuftelins, andderivatives thereof and mixtures thereof.
 63. A method according to anyof the claims 41-62, wherein the active enamel substance has a molecularweight of at the most about 120 kDa such as, e.g., at the most 100 kDa,90 kDa, 80 kDa, 70 kDa or 60 kDa as determined by SDS PAGEelectrophoresis.
 64. A method according to any of the claims 41-63,wherein the preparation of an active enamel substance contains a mixtureof active enamel substances with different molecular weights.
 65. Amethod according to any of the claims 41-64, wherein the preparation ofan active enamel substance comprises at least two substances selectedfrom the group consisting of amelogenins, proline-rich non-amelogenins,tuftelin, tuft proteins, serum proteins, salivary proteins,ameloblastin, sheathlin, and derivatives thereof.
 66. A method accordingto any of the claims 41-65, wherein the active enamel substance has amolecular weight of up to about 40,000.
 67. A method according to any ofthe claims 41-66, wherein the active enamel substance has a molecularweight of between about 5,000 and about 25,000.
 68. A method accordingto any of the claims 41-67, wherein the major part of the active enamelsubstance has a molecular weight of about 20 kDa.
 69. A method accordingto any of the claims 41-68, wherein the protein content of the activeenamel substance in the preparation is in a range of from about 0.005%w/w to 100% w/w such as, e.g., about 5-99% w/w, about 10-95% w/w, about15-90% w/w, about 20-90% w/w, about 30-90% w/w, about 40-85% w/w, about50-80% w/w, about 60-70% w/w, about 70-90% w/w, or about 80-90% w/w. 70.A method according to any of the claims 41-69, wherein thepharmaceutical composition further comprises a pharmaceuticallyacceptable excipient.
 71. A method according to claim 70, wherein thepharmaceutically acceptable excipient is phosphate buffer saline (PBS).72. A method according to claim 71, wherein the pharmaceuticalcomposition comprises 30 μg of enamel matrix protein and 1 ml ofphosphate buffer saline (PBS).